Enterohemorrhagic Escherichia coli vaccine

ABSTRACT

Compositions and methods for stimulating an immune response against a secreted enterohemorragic  Escherichia coli  (EHEC) antigen are disclosed. The compositions comprise EHEC cell culture supernatants.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 USC §119(e)(1) ofprovisional patent application Ser. No. 60/259,818, filed Jan. 4, 2001,which application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for elicitingan immune response in mammals against enterohemorragic Escherichia coli.In particular, the invention relates to the use of cell culturesupernatants for treating and preventing enterohemorragic E. colicolonization of mammals.

BACKGROUND OF THE INVENTION

Enterohemorragic Escherichia coli (EHEC), also called Shiga toxin E.coli (STEC) and vertotoxigenic E. coli (VTEC) are pathogenic bacteriathat cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome,kidney failure and death in humans. While many Shiga-liketoxin-producing EHEC strains are capable of causing disease in humans,those of serotype O157:H7 cause the majority of human illness. Thisorganism is able to colonize the large intestine of humans by a uniquemechanism in which a number of virulence determinants are delivered tohost cells via a type III secretion system, including the translocatedIntimin receptor, Tir (DeVinney et al., Infect. Immun. (1999) 67:2389).In particular, these pathogens secrete virulence determinants EspA, EspBand EspD that enable delivery of Tir into intestinal cell membranes. Tiris integrated into the host cell membrane where it serves as thereceptor for a bacterial outer membrane protein, Intimin. Tir-Intiminbinding attaches EHEC to the intestinal cell surface and triggers actincytoskeletal rearrangements beneath adherent EHEC that results inpedestal formation. EspA, EspB, Tir and Intimin are each essential forthe successful colonization of the intestine by EHEC.

Although EHEC colonize the intestine of ruminants and other mammals,they generally do not cause overt disease in these animals. However,contamination of meat and water by the EHEC serotype O157:H7(hereinafter, “EHEC O157:H7”) is responsible for about 50,000 cases ofEHEC O157:H7 infection in humans annually in the United States andCanada that result in approximately 500 deaths. In 1994, the economiccost associated with EHEC O157:H7 infection in humans was estimated tobe over 5 billion dollars annually.

The first documented EHEC O157:H7 outbreak traced to contaminated meatoccurred in 1982. Subsequently, it was demonstrated that healthyruminants including, but not limited to, cattle, dairy cows and sheep,could be infected with EHEC O157:H7. In fact, USDA reports indicate thatup to 50% of cattle are carriers of EHEC O157:H7 at some time duringtheir lifetime and, therefore, shed EHEC O157:H7 in their feces.

Because of the bulk processing of slaughtered cattle and the low numberof EHEC O157:H7 (10–100) necessary to infect a human, EHEC O157:H7colonization of healthy cattle remains a serious health problem. Toaddress this problem, research has focused on improved methods fordetecting and subsequently killing EHEC O157:H7 at slaughter, alteringthe diet of cattle to reduce the number of intestinal EHEC O157:H7 andimmunizing animals to prevent EHEC O157:H7 colonization. Recently, therecombinant production and use of EHEC O157:H7 proteins includingrecombinant EspA (International Publication No. WO 97/40063),recombinant TIR (International Publication No. WO 99/24576), recombinantEspB and recombinant Intimin (Li et al., Infec. Immun. (2000)68:5090–5095) have been described. However, production and purificationof recombinant proteins in amounts sufficient for use as antigens isboth difficult and expensive. At the present time, there is no effectivemethod for blocking EHEC O157:H7 colonization of cattle and othermammals and, thereby, for reducing shedding of EHEC into theenvironment.

Therefore, there is a need for new compositions and methods for treatingand preventing EHEC disease, as well as for reducing EHEC colonizationof mammals in order to reduce the incidence of health problemsassociated with EHEC-contaminated meat and water.

SUMMARY OF THE INVENTION

The present invention satisfies the above need by providing suchcompositions and methods. In particular, the methods of the presentinvention make use of a composition comprising a cell culturesupernatant (hereinafter “CCS”) derived from an EHEC culture to elicitan immune response against one or more EHEC secreted antigens, therebytreating and/or preventing EHEC infection and/or reducing EHECcolonization of the mammal. The compositions can be delivered with orwithout a coadministered adjuvant. In certain embodiments, EspA and Tircomprise at least 20% of the cell culture supernatant protein. The EHECculture supernatant may be derived from any EHEC serotype, but ispreferably obtained from a culture of EHEC O157:H7 and/or EHEC O157:NM(non-motile). The cell culture supernatant of the present invention iseasy and relatively inexpensive to prepare and is effective at doseregimens that have minimal toxicity.

EspA, EspB, Tir and Intimin are necessary for activation (A) of hostepithelial cell signal transduction pathways and for the intimateattachment (E) of EHEC to host epithelial cells. Therefore, withoutbeing bound by the following hypothesis, it is thought thatadministration of the CCS of the present invention to a mammalstimulates an immune response against one or more secreted antigens,such as EspA and Tir, that blocks attachment of the EHEC to intestinalepithelial cells.

Accordingly, it is an object of the present invention to provide avaccine effective to stimulate an immune response against EHEC secretedantigens, thereby treating and/or preventing EHEC disease in a mammal.

Another object is to provide a vaccine effective to reduce, preventand/or eliminate EHEC colonization of a ruminant or other mammal.

Another object is to reduce the number of animals shedding EHEC into theenvironment.

Another object is to reduce the number of EHEC shed into the environmentby an infected animal.

Another object is reduce the time during which EHEC are shed into theenvironment by an infected animal.

Another object is reduce EHEC contamination of the environment.

Another object is reduce EHEC contamination of meat and/or water.

Another object is to treat, prevent and/or reduce EHEC infections inhumans.

Another object is to provide a vaccine effective as an adjunct to otherbiological anti-EHEC agents.

Another object is to provide a vaccine effective as an adjunct tochemical anti-EHEC agents.

Another object is to provide a vaccine effective as an adjunct tobiologically engineered anti-EHEC agents.

Another object is to provide a vaccine effective as an adjunct tonucleic acid-based anti-EHEC agents.

Another object is to provide a vaccine effective as an adjunct torecombinant protein anti-EHEC agents.

Another object is to provide a vaccination schedule effective to reduceEHEC colonization of a ruminant.

Another object is to provide a vaccination schedule effective to reduceEHEC shedding by a ruminant.

Another object is to provide a vaccine effective to reduce EHEC O157colonization of cattle, such as colonization of EHEC O157:H7 and/or EHECO157:NM.

Another object is to provide a vaccine effective to prevent EHEC O157colonization of cattle, such as colonization of EHEC O157:H7 and/or EHECO157:NM.

Another object is to provide a vaccine effective to eliminate EHEC O157colonization of cattle, such as colonization of EHEC O157:H7 and/or EHECO157:NM.

Another object is to reduce the number of cattle shedding EHEC O157 intothe environment, such as shedding of EHEC O157:H7 and/or EHEC O157:NM.

Another object is to reduce the number of EHEC O157 shed into theenvironment by infected cattle, such as shedding of EHEC O157:H7 and/orEHEC O157:NM.

Another object is reduce the time during which EHEC O157 are shed intothe environment by infected cattle, such as shedding of EHEC O157:H7and/or EHEC O157:NM.

Another object is to provide a vaccine effective as an adjunct to otheranti-EHEC O157 agents.

Another object is to provide a vaccination schedule effective to reduceEHEC O157 colonization of cattle.

Another object is to provide a vaccination schedule effective to reduceEHEC O157 shedding by cattle.

Thus, in one embodiment, the invention is directed to a vaccinecomposition comprising an enterohemorragic Escherichia coli (EHEC) cellculture supernatant and an immunological adjuvant. In certainembodiments, the EHEC is EHEC O157:H7 and/or EHEC O157:NM. In additionalembodiments, the immunological adjuvant comprises an oil-in-wateremulsion, such as a mineral oil and dimethyldioctadecylammonium bromide.In yet additional embodiments, the immunological adjuvant is VSA3. TheVSA3 may be present at a concentration of about 20% to about 40% (v/v),such as at a concentration of 30% (v/v).

In still further embodiments, the vaccine composition further comprisesone or more recombinant or purified EHEC secreted antigens selected fromthe group consisting of EspA, EspB, EspD and Tir. In other embodiments,EspA+Tir comprise at least 20% of the cell protein present in thecomposition.

In further embodiments, the subject invention is directed to methods foreliciting an immunological response in a mammal against a secretedenterohemorragic Escherichia coli (EHEC) antigen. The method comprisesadministering to the mammal a therapeutically effective amount of acomposition comprising an EHEC cell culture supernatant. In certainembodiments, the EHEC is EHEC O157:H7 and/or EHEC O157:NM. In additionalembodiments, the mammal is a human or a ruminant, such as a bovinesubject. In yet further embodiments, the composition further comprisesan immunological adjuvant, such as an oil-in-water emulsion whichcomprises e.g., a mineral oil and dimethyldioctadecylammonium bromide.In additional embodiments, the adjuvant is VSA3. The compositions mayfurther comprise one or more recombinant or purified EHEC secretedantigens selected from the group consisting of EspA, EspB, EspD and Tir.In other embodiments, EspA+Tir comprise at least 20% of the cell proteinpresent in the composition.

In another embodiment, the invention is directed to a method foreliciting an immunological response in a ruminant against a secretedenterohemorragic Escherichia coli O157:H7 (EHEC O157:H7) antigen. Themethod comprises administering to the ruminant a therapeuticallyeffective amount of a composition comprising an EHEC O157:H7 cellculture supernatant and VSA3. In additional embodiments, VSA3 is presentin the composition at a concentration of about 20% to about 40% (v/v),such as at about 30% (v/v).

In still a further embodiment, the invention is directed to a method forreducing colonization of enterohemorragic Escherichia coli (EHEC) in aruminant comprising administering to the ruminant a therapeuticallyeffective amount of a composition comprising an EHEC cell culturesupernatant and an immunological adjuvant.

In yet another embodiment, the invention is directed to a method forreducing shedding of enterohemorragic Escherichia coli (EHEC) from aruminant comprising administering to the ruminant a therapeuticallyeffective amount of a composition comprising an EHEC cell culturesupernatant and an immunological adjuvant.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions, and aretherefore incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the electrophoretic profile of CCS proteins separated bypolyacrylamide gel electrophoresis.

FIG. 2 shows the electrophoretic profile of recombinant EspA, Tir, EspBand Intimin separated by polyacrylamide gel electrophoresis.

FIG. 3 shows fecal shedding of EHEC O157:H7 by cattle immunized with aCCS vaccine following EHEC O157:H7 challenge.

FIG. 4 depicts reactivation of fecal shedding of EHEC O157:H7 inpreviously infected cattle.

FIG. 5 shows the serological response to immunization with recombinantEspA+Tir vaccine and with recombinant EspB+Intimin vaccine.

FIG. 6 depicts fecal shedding of EHEC O157:H7 following immunizationwith recombinant EspA+Tir vaccine and with saline vaccine.

FIG. 7 shows the number of animals shedding E. coli O157:H7 on each dayof the vaccine trial described in Example 6. Bacteria were detected bydirect plating of fecal samples which had been resuspended in saline onSorbitol MaConkey agar supplemented with cefixime and tellurite. Solidbars, placebo group; hatched bars, EHEC vaccine group.

FIG. 8 shows an immunoblot analysis of sera from vaccinated animalsagainst EHEC secreted proteins. Each blot contains secreted proteinsfrom wild-type E. coli O157:H7 (EHEC), type III secretion mutant(ΔSepB), tir mutant (ΔTir) and a purified glutathione-s-transferase:Tirfusion protein (GST-Tir). Proteins were separated by SDS-10% PAGE andstained with Coomassie blue (A, upper left panel) or transferred tonitrocellulose and probed with representative sera from animals whichreceived 3 immunizations with each vaccine formulation (A, upperpanels). The lower four panels (B) were probed with sera from onerepresentative animal which received the EHEC vaccine, taken on days 0,21, 25 and 49 of the trial.

FIG. 9 shows the percentage of each group of animals shedding E. coliO157:H7(Panel A) and the total number of bacteria recovered (Panel B) oneach day of the trial described in Example 6. Bacteria were detected infeces by plating on Sorbitol MaConkey agar supplemented with cefiximeand tellurite following immunomagnetic enrichment as described in J. VanDonkersgoed et al., Can. Vet. J. (2001) 42:714. (A) Solid bars, placebo;hatched bars, EHEC vaccine; open bars, ΔTir vaccine. (B) ▪, placebogroup; ●, EHEC vaccine; ▴, ΔTir vaccine.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, and immunology, which are within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Vols. I, II and III, Second Edition (1989); Perbal, B., APractical Guide to Molecular Cloning (1984); the series, Methods InEnzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); andHandbook of Experimental Immunology, Vols. I–IV (D. M. Weir and C. C.Blackwell eds., 1986, Blackwell Scientific Publications).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “an EHEC bacterium ” includes a mixture of two or more suchbacteria, and the like.

As used herein, the term EHEC “cell culture supernatant” or “CCS” refersto a supernatant derived from a cell culture of one or more EHECserotypes, which supernatant is substantially free of EHEC bacterialcells or the lysate of such cells, and which contains a mixture of EHECantigens that have been secreted into the growth media. Generally, anEHEC “CCS” will contain at least the secreted antigens EspA, EspB, EspDand Tir, and fragments or aggregates thereof. The CCS of the presentinvention may also include other secreted proteins, such as EspF andMAP, one or both of Shiga toxins 1 and 2, as well as EspP which is anapproximately 100 kDa protein which is not secreted by the type IIIsystem. The proteins can be present in a native form, or a denatured ordegraded form, so long as the CCS still functions to stimulate an immuneresponse in the host subject such that EHEC disease is lessened orprevented, and/or colonization of EHEC is lessened or suppressed. Insome instances, a CCS may be supplemented with additional recombinant orpurified secreted antigens, such as with additional EspA, EspB, EspDand/or Tir, as well as with any of the other secreted proteins, and mayalso be supplemented with Intimin. In certain embodiments, EspA+Tir willcomprise at least 20% of the cell culture supernatant protein.

As used herein, a “recombinant” EHEC secreted protein, such as rEspA,rEspB, rEspD and rTir, as well as the “recombinant Intimin”, refers tothe full-length polypeptide sequence, fragments of the referencesequence or substitutions, deletions and/or additions to the referencesequence, so long as the proteins retain at least one specific epitopeor activity. Generally, analogs of the reference sequence will displayat least about 50% sequence identity, preferably at least about 75% to85% sequence identity, and even more preferably about 90% to 95% or moresequence identity, to the full-length reference sequence. See, e.g.,GenBank Accession Nos. AE005594, AE005595, AP002566, AE005174,NC_(—)002695, NC_(—)002655 for the complete sequence of the E. coliO157:H7 genome, which includes the sequences of the various O157:H7secreted proteins. See, e.g., International Publication No. WO 97/40063,as well as GenBank Accession Nos. Y13068, U80908, U5681, Z54352,AJ225021, AJ225020, AJ225019, AJ225018, AJ225017, AJ225016, AJ225015,AF022236 and AF200363 for the nucleotide and amino acid sequences ofEspA from a number of E. coli serotypes. See, e.g., InternationalPublication No. WO 99/24576, as well as GenBank Accession Nos. AF125993,AF132728, AF045568, AF022236, AF70067, AF070068, AF013122, AF200363,AF113597, AF070069, AB036053, AB026719, U5904 and U59502, for thenucleotide and amino acid sequences of Tir from a number of E. coliserotypes. See, e.g., GenBank Accession Nos. U32312, U38618, U59503,U66102, AF081183, AF081182, AF130315, AF339751, AJ308551, AF301015,AF329681, AF319597, AJ275089–AJ275113 for the nucleotide and amino acidsequences of Intimin from a number of E. coli serotypes. See, e.g.,GenBank Accession Nos. U80796, U65681, Y13068, Y13859, X96953, X99670,X96953, Z21555, AF254454, AF254455, AF254456, AF254457, AF054421,AF059713, AF144008, AF144009 for the nucleotide and amino acid sequencesof EspB from a number of E. coli serotypes. See, e.g., GenBank AccessionNos. Y13068, Y13859, Y17875, Y17874, Y09228, U65681, AF054421 andAF064683, for the nucleotide and amino acid sequences of EspD from anumber of E. coli serotypes.

“Homology” refers to the percent similarity between two polynucleotideor two polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 80%–85%, preferably at least about 90%, and most preferablyat least about 95%–98% sequence similarity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

Percent sequence identity can be determined by a direct comparison ofthe sequence information between two molecules by aligning thesequences, counting the exact number of matches between the two alignedsequences, dividing by the length of the shorter sequence, andmultiplying the result by 100. Readily available computer programs canbe used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlasof Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353–358,National biomedical Research Foundation, Washington, DC, which adaptsthe local homology algorithm of Smith and Waterman (1981) Advances inAppl. Math. 2:482–489 for peptide analysis. Programs for determiningnucleotide sequence identity are available in the Wisconsin SequenceAnalysis Package, Version 8 (available from Genetics Computer Group,Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, whichalso rely on the Smith and Waterman algorithm. These programs arereadily utilized with the default parameters recommended by themanufacturer and described in the Wisconsin Sequence Analysis Packagereferred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

As used herein, “vaccine” refers to a CCS composition that serves tostimulate an immune response to an EHEC antigen, such as a type IIIsecreted EHEC antigen, therein. The immune response need not providecomplete protection and/or treatment against EHEC infection or againstcolonization and shedding of EHEC. Even partial protection againstcolonization and shedding of EHEC bacteria will find use herein asshedding and contaminated meat production will still be reduced. In somecases, a vaccine will include an immunological adjuvant in order toenhance the immune response. The term “adjuvant” refers to an agentwhich acts in a nonspecific manner to increase an immune response to aparticular antigen or combination of antigens, thus reducing thequantity of antigen necessary in any given vaccine, and/or the frequencyof injection necessary in order to generate an adequate immune responseto the antigen of interest. See, e.g., A. C. Allison J.Reticuloendothel. Soc. (1979) 26:619–630. Such adjuvants are describedfurther below.

As used herein, “colonization” refers to the presence of EHEC in theintestinal tract of a mammal, such as a ruminant.

As used herein, “shedding” refers to the presence of EHEC in feces.

As used herein, “therapeutic amount”, “effective amount” and “amounteffective to” refer to an amount of vaccine effective to elicit animmune response against a secreted antigen present in the CCS, therebyreducing or preventing EHEC disease, and/or EHEC colonization of amammal such as a ruminant; and/or reducing the number of animalsshedding EHEC; and/or reducing the number of EHEC shed by an animal;and/or, reducing the time period of EHEC shedding by an animal.

As used herein, “immunization” or “immunize” refers to administration ofCCS, with or without additional recombinant or purified EHEC antigenssuch as EspA, Tir, EspB, EspD, and/or Intimin, in an amount effective tostimulate the immune system of the animal to which the CCS isadministered, to elicit an immunological response against one or more ofthe secreted antigens present in the CCS.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite.”

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells,directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. Preferably, the host will displayeither a therapeutic or protective immunological response such that EHECdisease is lessened and/or prevented; resistance of the intestine tocolonization with EHEC is imparted; the number of animals shedding EHECis reduced; the number of EHEC shed by an animal is reduced; and/or thetime period of EHEC shedding by an animal is reduced.

The terms “immunogenic” protein or polypeptide refer to an amino acidsequence which elicits an immunological response as described above. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the particular EHEC protein in question, analogsthereof, aggregates, or immunogenic fragments thereof. By “immunogenicfragment” is meant a fragment of a secreted EHEC protein which includesone or more epitopes and thus elicits the immunological responsedescribed above. Such fragments can be identified using any number ofepitope mapping techniques, well known in the art. See, e.g., EpitopeMapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E.Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linearepitopes may be determined by e.g., concurrently synthesizing largenumbers of peptides on solid supports, the peptides corresponding toportions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998–4002;Geysen et al. (1986) Molec. Immunol. 23:709–715, all incorporated hereinby reference in their entireties. Similarly, conformational epitopes arereadily identified by determining spatial conformation of amino acidssuch as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra.Antigenic regions of proteins can also be identified using standardantigenicity and hydropathy plots, such as those calculated using, e.g.,the Omiga version 1.0 software program available from the OxfordMolecular Group. This computer program employs the Hopp/Woods method,Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824–3828 fordetermining antigenicity profiles, and the Kyte-Doolittle technique,Kyte et al., J. Mol. Biol. (1982) 157:105–132 for hydropathy plots.

Immunogenic fragments, for purposes of the present invention, willusually include at least about 3 amino acids, preferably at least about5 amino acids, more preferably at least about 10–15 amino acids, andmost preferably 25 or more amino acids, of the parent EHEC secretedprotein molecule. There is no critical upper limit to the length of thefragment, which may comprise nearly the full-length of the proteinsequence, or even a fusion protein comprising two or more epitopes ofthe particular EHEC secreted protein.

“Native” proteins or polypeptides refer to proteins or polypeptidesisolated from the source in which the proteins naturally occur.“Recombinant” polypeptides refer to polypeptides produced by recombinantDNA techniques; i.e., produced from cells transformed by an exogenousDNA construct encoding the desired polypeptide. “Synthetic” polypeptidesare those prepared by chemical synthesis.

The term “treatment” as used herein refers to either (i) the preventionof infection or reinfection (prophylaxis), or (ii) the reduction orelimination of symptoms of the disease of interest (therapy).

By “mammalian subject” is meant any member of the class Mammalia,including humans and all other mammary gland possessing animals (bothmale and female), such as ruminants, including, but not limited to,bovine, porcine and Ovis (sheep and goats) species. The term does notdenote a particular age. Thus, adults, newborns, and fetuses areintended to be covered.

B. General Methods

Central to the present invention is the discovery that cell culturesupernatants derived from EHEC cultures which contain EHEC secretedantigens, produce an immune response in animals to which they areadministered and thereby provide protection against EHEC infection, suchas protection against colonization. In certain embodiments, thecompositions comprise a mixture of EHEC secreted antigens, including butnot limited to EspA, EspB, EspD and/or Tir. The CCS of the presentinvention may also include other secreted proteins, such as EspF andMAP, one or both of Shiga toxins 1 and 2, as well as EspP which is anapproximately 100 kDa protein which is not secreted by the type IIIsystem. In other embodiments, the CCS is supplemented with additionalrecombinant or purified EHEC antigens, such as with additional EspA,EspB, EspD, Tir, Intimin, and the like. In certain embodiments, EspA+Tircomprise at least 20% of the cell culture supernatant protein. Thecompositions can comprise cell culture supernatants and additionaladjuvants from more than one EHEC serotype to provide protection againstmultiple EHEC organisms. Moreover, a pharmaceutically acceptableadjuvant may be administered with the cell culture supernatant. Thecompositions are administered in an amount effective to elicit an immuneresponse to one or more of the secreted antigens, thereby reducing oreliminating EHEC infection. In some instances, EHEC colonization of theanimal is reduced or eliminated. In preferred embodiments, the animal isa cow or a sheep or other ruminant. In particularly preferredembodiments, the cell culture supernatant is derived from a cell cultureof EHEC O157:H7 or EHEC O157:NM.

Immunization with CCS stimulates the immune system of the immunizedanimal to produce antibodies against one or more secreted EHEC antigens,such as EspA, EspB, EspD and Tir, that block EHEC attachment tointestinal epithelial cells, interfere with EHEC colonization and,thereby, reduce EHEC shedding by the animal. This reduction in EHECshedding results in a reduction in EHEC contamination of food and waterand a reduction in EHEC-caused disease in humans. Moreover, theunexpected and surprising ability of CCS immunization to prevent, reduceand eliminate EHEC colonization and shedding by cattle addresses along-felt unfulfilled need in the medical arts, and provides animportant benefit for humans.

Additionally, the CCS of the present invention can be used to treat orprevent EHEC infections in other mammals such as humans. If used inhumans, the CCS can be produced from a mutated EHEC which has beenengineered to knock out one or both of the Shiga toxins 1 and 2 in orderto reduce toxicity.

As explained above, the therapeutic effectiveness of CCS can beincreased by adding thereto one or more of the secreted antigens inrecombinant or purified form, such as by adding recombinant or purifiedEspA, EspB, EspD, Tir, and the like, fragments thereof and/or analogsthereof. Intimin may also be added. Other methods to increase thetherapeutic effectiveness of CCS include, but are not limited to,complexing the CCS to natural or synthetic carriers and administeringthe CCS, before, at the same time as, or after another anti-EHEC agent.Such agents include, but are not limited to, biological, biologicallyengineered, chemical, nucleic acid based and recombinant proteinanti-EHEC agents.

CCS from pathogenic bacteria, other than serotypes of EHEC, that requireproteins such as EspA and Tir to colonize a host, can also be used tostimulate the immune system of an animal to produce antibodies againstsecreted EHEC antigens that reduce bacterial binding to intestinalepithelial cells of the animal. These bacterial species include, but arenot limited to Citrotobacter rodentium.

The CCS for use herein may be obtained from cultures of any EHECserotype, including, without limitation, EHEC serotypes from serogroupsO157, O158, O5, O8, O18, O26, O45, O48, O52, O55, O75, O76, O78,O84,O91, O103, O104, O111, O113, O114, O116, O118, O119, O121, O125,O28, O145, O146, O163, O165. Such EHEC serotypes are readily obtainedfrom sera of infected animals. Methods for isolated EHEC are well knownin the art. See, e.g., Elder et al., Proc. Natl. Acad. Sci. USA (2000)97:2999; Van Donkersgoed et al., Can. Vet. J. (1999) 40:332; VanDonkersgoed et al., Can. Vet. J. (2001) 42:714. Generally, such methodsentail direct plating on sorbitol MacConkey agar supplemented withcefixime and tellurite or immunomagnetic enrichment followed by platingon the same media. Moreover, CCS may be obtained from EHEC serotypesthat have been genetically engineered to knock-out expression of Shigatoxins 1 and/or 2, in order to reduce toxicity.

Generally, CCS is produced by culturing EHEC bacteria in a suitablemedium, under conditions that favor type III antigen secretion. Suitablemedia and conditions for culturing EHEC bacteria are known in the artand described in e.g., U.S. Pat. Nos. 6,136,554 and 6,165,743(incorporated herein by reference in their entireties), as well as in Liet al., Infec. Immun. (2000) 68:5090–5095; Fey et al., Emerg. Infect.Dis. (2000) Volume 6. A particularly preferable method of obtaining CCSis by first growing organisms in Luria-Bertani (LB) medium for a periodof about 8 to 48 hours, preferably about 12 to 24 hours, and dilutingthis culture about 1:5 to 1:50, preferably 1:5 to 1:25, more preferablyabout 1:10, into M-9 minimal medium supplemented with 20–100 mM NaHCO₃,preferably 30–50 mM, most preferably about 44 mM NaHCO₃, 4–20 mM MgSO₄,preferably 5–10 mM and most preferably about 8 mM MgSO₄, 0.1 to 1.5%glucose, preferable 0.2 to 1%, most preferably 0.4% glucose and 0.05 to0.5% Casamino Acids, preferably 0.07 to 0.2%, most preferably about 0.1%Casamino Acids. Cultures are generally maintained at about 37 degrees C.in 2–10% CO₂, preferably about 5% CO₂, to an optical density of about600 nm of 0.7 to 0.8. Whole cells are then removed by centrifugation andthe supernatant can be concentrated, e.g., 10–1000 fold or more, such as100-fold, using dialysis, ultrafiltration and the like. Total protein iseasily determined using methods well known in the art.

As explained above, the CCS can be supplemented with additional EHECsecreted proteins, such as EspA, EspB, EspD and/or Tir. Intimin may alsobe added. These proteins can be produced recombinantly using techniqueswell known in the art. See, e.g., International Publication Nos. WO97/40063 and WO 99/24576 for a description of the production ofrepresentative recombinant EHEC secreted proteins. In particular, thesequences for EspA, EspB, EspD, Tir and Intimin from various serotypesare known and described. See, e.g., GenBank Accession Nos. AE005594,AE005595, AP002566, AE005174, NC_(—)002695, NC _(—)002655 for thecomplete sequence of the E. coli O157:H7 genome, which includes thesequences of the various O157:H7 secreted proteins. See, e.g.,International Publication No. WO 97/40063, as well as GenBank AccessionNos. Y13068, U80908, U5681, Z54352, AJ225021, AJ225020, AJ225019,AJ225018, AJ225017, AJ225016, AJ225015, AF022236 and AF200363 for thenucleotide and amino acid sequences of EspA from a number of E. coliserotypes. See, e.g., International Publication No. WO 99/24576, as wellas GenBank Accession Nos. AF125993, AF132728, AF045568, AF022236,AF70067, AF070068, AF013122, AF200363, AF113597, AF070069, AB036053,AB026719, U5904 and U59502, for the nucleotide and amino acid sequencesof Tir from a number of E. coli serotypes. See, e.g., GenBank AccessionNos. U32312, U38618, U59503, U66102, AF081183, AF081182, AF130315,AF339751, AJ308551, AF301015, AF329681, AF319597, AJ275089–AJ275113 forthe nucleotide and amino acid sequences of Intimin from a number of E.coli serotypes. See, e.g., GenBank Accession Nos. U80796, U65681,Y13068, Y13859, X96953, X99670, X96953, Z21555, AF254454, AF254455,AF254456, AF254457, AF054421, AF059713, AF144008, AF144009 for thenucleotide and amino acid sequences of EspB from a number of E. coliserotypes. See, e.g., GenBank Accession Nos. Y13068, Y13859, Y17875,Y17874, Y09228, U65681, AF054421 and AF064683, for the nucleotide andamino acid sequences of EspD from a number of E. coli serotypes.

These sequences can be used to design oligonucleotide probes and used toscreen genomic or cDNA libraries for genes from other E. coli serotypes.The basic strategies for preparing oligonucleotide probes and DNAlibraries, as well as their screening by nucleic acid hybridization, arewell known to those of ordinary skill in the art. See, e.g., DNACloning: Vol. I, supra; Nucleic Acid Hybridization, supra;Oligonucleotide Synthesis, supra; Sambrook et al., supra. Once a clonefrom the screened library has been identified by positive hybridization,it can be confirmed by restriction enzyme analysis and DNA sequencingthat the particular library insert contains a type III gene or a homologthereof. The genes can then be further isolated using standardtechniques and, if desired, PCR approaches or restriction enzymesemployed to delete portions of the full-length sequence.

Similarly, genes can be isolated directly from bacteria using knowntechniques, such as phenol extraction and the sequence furthermanipulated to produce any desired alterations. See, e.g., Sambrook etal., supra, for a description of techniques used to obtain and isolateDNA. Alternatively, DNA sequences encoding the proteins of interest canbe prepared synthetically rather than cloned. The DNA sequences can bedesigned with the appropriate codons for the particular amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge(1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay etal. (1984) J. Biol. Chem. 259:6311.

Once coding sequences for the desired proteins have been prepared orisolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B.Perbal, supra.

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. Leader sequences can be removed by the hostin post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;4,425,437; 4,338,397.

Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Regulatory sequences are known to those of skill inthe art, and examples include those which cause the expression of a geneto be turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements may also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector, such as thecloning vectors described above. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

In some cases it may be necessary to modify the coding sequence so thatit may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It may also bedesirable to produce mutants or analogs of the protein. Mutants oranalogs may be prepared by the deletion of a portion of the sequenceencoding the protein, by insertion of a sequence, and/or by substitutionof one or more nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are describedin, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Similarly,bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcusspp., will find use with the present expression constructs. Yeast hostsuseful in the present invention include inter alia, Saccharomycescerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha,Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii,Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by culturing host cells transformedby an expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. The selection of the appropriate growthconditions and recovery methods are within the skill of the art.

The proteins of the present invention may also be produced by chemicalsynthesis such as solid phase peptide synthesis, using known amino acidsequences or amino acid sequences derived from the DNA sequence of thegenes of interest. Such methods are known to those skilled in the art.See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis,2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and R.B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E.Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp.3–254, for solid phase peptide synthesis techniques; and M. Bodansky,Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E.Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, supra, Vol. 1, for classical solution synthesis. Chemicalsynthesis of peptides may be preferable if a small fragment of theantigen in question is capable of raising an immunological response inthe subject of interest.

Once the above cell culture supernatants and, if desired, additionalrecombinant and/or purified proteins are produced, they are formulatedinto compositions for delivery to a mammalian subject. The CCS isadministered alone, or mixed with a pharmaceutically acceptable vehicleor excipient. Suitable vehicles are, for example, water, saline,dextrose, glycerol, ethanol, or the like, and combinations thereof. Inaddition, the vehicle may contain minor amounts of auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents, or adjuvantsin the case of vaccine compositions, which enhance the effectiveness ofthe vaccine. Suitable adjuvants are described further below. Thecompositions of the present invention can also include ancillarysubstances, such as pharmacological agents, cytokines, or otherbiological response modifiers.

As explained above, vaccine compositions of the present invention mayinclude adjuvants to further increase the immunogenicity of one or moreof the EHEC antigens. Such adjuvants include any compound or compoundsthat act to increase an immune response to an EHEC antigen orcombination of antigens, thus reducing the quantity of antigen necessaryin the vaccine, and/or the frequency of injection necessary in order togenerate an adequate immune response. Adjuvants may include for example,emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such asaluminum hydroxide, chitosan-based adjuvants, and any of the varioussaponins, oils, and other substances known in the art, such as AMPHIGEN™(AMPHIGEN™ comprises de-oiled lecithin dissolved in an oil, usuallylight liquid paraffin. In vaccine preparations AMPHIGEN™ is dispersed inan aqueous solution or suspension of the immunizing antigen as anoil-in-water emulsion), LPS, bacterial cell wall extracts, bacterialDNA, synthetic oligonucleotides and combinations thereof (Schijns etal., Curr. Opi. Immunol. (2000) 12:456), Mycobacterial phlei (M. phlei}cell wall extract (MCWE) (U.S. Pat. No. 4,744,984), M. phlei DNA(M-DNA), M-DNA-M phlei cell wall complex (MCC). For example, compoundswhich may serve as emulsifiers herein include natural and syntheticemulsifying agents, as well as anionic, cationic and nonionic compounds.Among the synthetic compounds, anionic emulsifying agents include, forexample, the potassium, sodium and ammonium salts of lauric and oleicacid, the calcium, magnesium and aluminum salts of fatty acids (i.e.,metallic soaps), and organic sulfonates such as sodium lauryl sulfate.Synthetic cationic agents include, for example, cetyltrimethylammoniumbromide, while synthetic nonionic agents are exemplified by glycerylesters (e.g., glyceryl monostearate), polyoxyethylene glycol esters andethers, and the sorbitan fatty acid esters (e.g., sorbitanmonopalmitate) and their polyoxyethylene derivatives (e.g.,polyoxyethylene sorbitan monopalmitate). Natural emulsifying agentsinclude acacia, gelatin, lecithin and cholesterol.

Other suitable adjuvants can be formed with an oil component, such as asingle oil, a mixture of oils, a water-in-oil emulsion, or anoil-in-water emulsion. The oil may be a mineral oil, a vegetable oil, oran animal oil. Mineral oil, or oil-in-water emulsions in which the oilcomponent is mineral oil are preferred. In this regard, a “mineral oil”is defined herein as a mixture of liquid hydrocarbons obtained frompetrolatum via a distillation technique; the term is synonymous with“liquid paraffin,” “liquid petrolatum” and “white mineral oil.” The termis also intended to include “light mineral oil,” i.e., an oil which issimilarly obtained by distillation of petrolatum, but which has aslightly lower specific gravity than white mineral oil. See, e.g.,Remington's Pharmaceutical Sciences, supra. A particularly preferred oilcomponent is the oil-in-water emulsion sold under the trade name ofEMULSIGEN PLUS™ (comprising a light mineral oil as well as 0.05%formalin, and 30 mcg/mL gentamicin as preservatives), available from MVPLaboratories, Ralston, Nebr. Suitable animal oils include, for example,cod liver oil, halibut oil, menhaden oil, orange roughy oil and sharkliver oil, all of which are available commercially. Suitable vegetableoils, include, without limitation, canola oil, almond oil, cottonseedoil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybeanoil, and the like.

Alternatively, a number of aliphatic nitrogenous bases can be used asadjuvants with the vaccine formulations. For example, known immunologicadjuvants include amines, quaternary ammonium compounds, guanidines,benzamidines and thiouroniums (Gall, D. (1966) Immunology 11:369–386).Specific compounds include dimethyldioctadecylammonium bromide (DDA)(available from Kodak) andN,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine (“avridine”). Theuse of DDA as an immunologic adjuvant has been described; see, e.g., theKodak Laboratory Chemicals Bulletin 56(1):1–5 (1986); Adv. Drug Deliv.Rev. 5(3):163–187 (1990); j. controlled Release 7:123–132 (1988); Clin.Exp. Immunol. 78(2):256–262 (1989); J. Immunol. Methods 97(2):159–164(1987); Immunology 58(2):245–250 (1986); and Int. Arch. Allergy Appl.Immunol. 68(3):201–208 (1982). Avridine is also a well-known adjuvant.See, e.g., U.S. Pat. No. 4,310,550 to Wolff, III et al., which describesthe use of N,N-higher alkyl-N′,N′-bis(2-hydroxyethyl)propane diamines ingeneral, and avridine in particular, as vaccine adjuvants. U.S. Pat. No.5,151,267 to Babiuk, and Babiuk et al. (1986) Virology 159:57–66, alsorelate to the use of avridine as a vaccine adjuvant.

Particularly preferred for use herein is an adjuvant known as “VSA3”which is a modified form of the EMULSIGEN PLUS™ adjuvant which includesDDA (see, U.S. Pat. No. 5,951,988, incorporated herein by reference inits entirety).

CCS vaccine compositions can be prepared by uniformly and intimatelybringing into association the CCS preparations and the adjuvant usingtechniques well known to those skilled in the art including, but notlimited to, mixing, sonication and microfluidation. The adjuvant willpreferably comprise about 10 to 50% (v/v) of the vaccine, morepreferably about 20 to 40% (v/v) and most preferably about 20 to 30% or35% (v/v), or any integer within these ranges.

The compositions of the present invention are normally prepared asinjectables, either as liquid solutions or suspensions, or as solidforms which are suitable for solution or suspension in liquid vehiclesprior to injection. The preparation may also be prepared in solid form,emulsified or the active ingredient encapsulated in liposome vehicles orother particulate carriers used for sustained delivery. For example, thevaccine may be in the form of an oil emulsion, water in oil emulsion,water-in-oil-in-water emulsion, site-specific emulsion, long-residenceemulsion, sticky-emulsion, microemulsion, nanoemulsion, liposome,microparticle, microsphere, nanosphere, nanoparticle and various naturalor synthetic polymers, such as nonresorbable impermeable polymers suchas ethylenevinyl acetate copolymers and Hytrel® copolymers, swellablepolymers such as hydrogels, or resorbable polymers such as collagen andcertain polyacids or polyesters such as those used to make resorbablesutures, that allow for sustained release of the vaccine.

Furthermore, the polypeptides may be formulated into compositions ineither neutral or salt forms. Pharmaceutically acceptable salts includethe acid addition salts (formed with the free amino groups of the activepolypeptides) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or organic acids such asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from freecarboxyl groups may also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18thedition, 1990.

The composition is formulated to contain an effective amount of secretedEHEC antigen, the exact amount being readily determined by one skilledin the art, wherein the amount depends on the animal to be treated andthe capacity of the animal's immune system to synthesize antibodies. Thecomposition or formulation to be administered will contain a quantity ofone or more secreted EHEC antigens adequate to achieve the desired statein the subject being treated. For purposes of the present invention, atherapeutically effective amount of a vaccine comprising CCS with orwithout added recombinant and/or purified secreted EHEC antigens,contains about 0.05 to 1500 μg secreted EHEC protein, preferably about10 to 1000 μg secreted EHEC protein, more preferably about 30 to 500 μgand most preferably about 40 to 300 μg, or any integer between thesevalues. EspA+Tir, as well as other EHEC antigens, may comprise about 10%to 50% of total CCS protein, such as about 15% to 40% and mostpreferably about 15% to 25%. If supplemented with rEspA+rTir, thevaccine may contain about 5 to 500 μg of protein, more preferably about10 to 250 μg and most preferably about 20 to 125 μg.

Routes of administration include, but are not limited to, oral, topical,subcutaneous, intramuscular, intravenous, subcutaneous, intradermal,transdermal and subdermal. Depending on the route of administration, thevolume per dose is preferably about 0.001 to 10 ml, more preferablyabout 0.01 to 5 ml, and most preferably about 0.1 to 3 ml. Vaccine canbe administered in a single dose treatment or in multiple dosetreatments (boosts) on a schedule and over a time period appropriate tothe age, weight and condition of the subject, the particular vaccineformulation used, and the route of administration.

Any suitable pharmaceutical delivery means may be employed to deliverthe compositions to the vertebrate subject. For example, conventionalneedle syringes, spring or compressed gas (air) injectors (U.S. Pat. No.1,605,763 to Smoot; U.S. Pat. No. 3,788,315 to Laurens; U.S. Pat. No.3,853,125 to Clark et al.; U.S. Pat. No. 4,596,556 to Morrow et al.; andU.S. Pat. No. 5,062,830 to Dunlap), liquid jet injectors (U.S. Pat. No.2,754,818 to Scherer; U.S. Pat. No. 3,330,276 to Gordon; and U.S. Pat.No. 4,518,385 to Lindmayer et al.), and particle injectors (U.S. Pat.No. 5,149,655 to McCabe et al. and U.S. Pat. No. 5,204,253 to Sanford etal.) are all appropriate for delivery of the compositions.

If a jet injector is used, a single jet of the liquid vaccinecomposition is ejected under high pressure and velocity, e.g., 1200–1400PSI, thereby creating an opening in the skin and penetrating to depthssuitable for immunization.

The following examples will serve to further illustrate the presentinvention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

C. Experimental

EXAMPLE 1

Preparation of Cell Culture Supernatant (CCS)

Wild type EHEC O157:H7 were grown under conditions to maximize thesynthesis of CCS proteins (Li et al., Infect. Immun. (2000) 68:5090).Briefly, an overnight standing culture of EHEC O157:H7 was grown inLuria-Bertani (LB) medium overnight at 37° C. (±5% CO₂). The culture wasdiluted 1:10 in M-9 minimal medium supplemented with 0.1% CasaminoAcids, 0.4% glucose, 8 mM MgSO₄ and 44 mM NaHCO₃. Cultures were grownstanding at 37° C. in 5% CO₂ to an optical density at 600 nm of 0.7 to0.8 (6–8 h). Bacteria were removed by centrifugation at 8000 rpm for 20min at 4° C. The supernatant was concentrated 100 fold byultrafiltration and total protein was determined by the bicinchoninicacid protein assay method.

FIG. 1 shows molecular weight markers (lane 1) and a typical CCS proteinprofile obtained by electrophoresis of CCS in a SDS-10% polyacrylamidegel (SDS-PAGE) followed by Coomassie blue staining (lane 2). Thepositions of EspA (25 kD), EspB/EspD (40 kD), undegraded Tir (70 kD) anddegraded Tir (55 kD) are indicated. As determined by densitometricanalysis using an HP Scanjet 5100C and the ID software program fromAdvance American Biotechnology (Fullerton, Calif., USA), EspA was about5% undegraded Tir about 20% and degraded Tir about 6% of the totalprotein. However, the percentages of proteins determined bydensitometric analysis of Coomassie blue stained SDS-polyacrylamide gelsis not exact due to variations in background staining, variations in theuptake of the Coomassie blue stain, variations in the density of thebands, and other factors known to those skilled in the art.

EXAMPLE 2

Preparation of Recombinant Proteins

The genes coding for EspA, EspB, Intimin and Tir were isolated (Li etal., Infect. Immun. (2000) 68:5090). A clinical isolate of EHEC O157:H7was used as the source of DNA. EspA, EspB, Tir, and the region of eaeencoding the 280 carboxyl-terminal amino acids of Intimin were amplifiedfrom chromosomal DNA using PCR to introduce unique restriction sites,followed by cloning into appropriate plasmids. The resulting plasmidswere cleaved and ligated to create histidine-tagged fusions. Plasmidswere electrocuted into an expression strain of E. coli and the E. coliwere propagated (Ngeleka et al., Infect. Immun. (1996) 64:3118). Geneexpression was driven using the Tac promoter following IPTG(isopropyl-β-D-thiogalactopyranoside) induction. Bacteria were pelleted,resuspended in Tris-buffered saline and lysed by sonication. The lysatewas centrifuged to remove insoluble material and the histidine-taggedproteins were purified by passage through a solid-phase nickel affinitychromatography column that specifically binds proteins containing thehistidine tag. All recombinant protein preparations were stored at −20°C. until use.

The purity of the recombinant proteins was assessed by SDS-PAGE on 10%gels followed by Coomassie blue staining. Typical gel profiles of thechromatographically purified recombinant (r) proteins are shown in FIG.2. rEspA (lane 2) rEspB (lane 3) and rIntimin (lane 4), were recoveredin relatively pure form, but rTir (lane 5) was subject to somedegradation.

EXAMPLE 3

Vaccine Formulation and Delivery

Vaccines were formulated by mixing CCS or rEspA+rTir in 2 ml of acarrier containing from 30 to 40% of an adjuvant. Vaccines weredelivered subcutaneously. Animals were immunized on day 1 and again at a3–4 week intervals (boost). Serum samples were obtained prior to thefirst immunization, at the time of each boost and at the end of theexperiment.

The serological response to immunization was determined using anenzyme-linked immunosorbent assay (ELISA). One hundred μl of rEspA (0.16μg/well), rTir (0.1 μg/well), rEspB (0.24 μg/well) and rIntimin (0.187μg/well) were used to coat the wells in microtiter plates and the plateswere incubated overnight at 4° C. The wells were washed 3×, blocked with0.5% nonfat dried milk in phosphate-buffered saline. Serial dilutions ofsera were added to each well and incubated for 2 h at 37° C. The wellswere washed and blocked and 100 μl of peroxidase-conjugated rabbitanti-bovine immunoglobulin G antibodies (1:5000) were added to each wellfor 1 h at 37° C. The wells were washed and plates were read at awavelength of 492 nm.

EXAMPLE 4

Experimental Animals

Cattle, between the ages of 8 and 12 months, were purchased from localranchers. Fecal samples were obtained daily from each animal for 14days. The number of EHEC O157:H7 in the fecal samples was determined byplating on Rainbow Agar. The plates were incubated at 37° C. for 2 daysand black colonies were enumerated. Growth was scored from 0–5. Animalshaving a score of 0 (no EHEC O157:H7) were used in all experiments.

EXAMPLE 5

Animal Colonization Model

A model for EHEC O157:H7 colonization of cattle, wherein the infectionwas sustained for >2 months, was developed using a dose-titrationprotocol.

EHEC O157:H7 were grown as in Example 1. Twenty-four cattle were dividedinto 3 groups of 8 animals each. Group 1 received 10^(6,) Group 2 10⁸and Group 3 10¹⁰ CFU of EHEC O157:H7 by oral-gastric intubation in avolume of 50 ml on day 0.

To monitor shedding, fecal material was collected on days 1 through 14.The fecal material was weighed, suspended in sterile saline andinoculated into culture media. Culture density was determined as inExample 1.

As shown in FIG. 3, there was no significant difference between numbersof EHEC O157:H7 shed by Group 2 (10⁸ CFU) and Group 3 (10¹⁰ CFU) cattle.Group 2 cattle shed the most EHEC O157:H7 on each of the 14 days. Thenumber of EHEC O157,.H7 shed by Group 2 cattle reached a maximum on day6 and declined to zero by day 14.

Animals shedding EHEC O157:H7 (hereinafter, “positive”) were kept anadditional 40 days during which time the number of EHEC O157:H7 sheddecreased to an undetectable level. The shedding of EHEC O157:H7 bypreviously positive animals (hereinafter, “carriers”) was reactivated bywithholding feed for 24 hours and vaccinating withcommercially-available clostridial or H. somnus vaccines. As shown inFIG. 4, the number of carrier animals shedding EHEC O157:H7 reached amaximum of approximately 50% on days 6 and 7 post-reactivation anddeclined to zero by day 15.

As a dose of 10⁸ CFU produced a detectable number of shed EHEC O157:H7during the 14 days post-infection (FIG. 3) and resulted in persistentlyinfected animals (FIG. 4), this dose was used as the challenge dose insubsequent experiments.

EXAMPLE 6

Protective Capacity of CCS

To test the vaccine potential of secreted proteins, CCS was mixed withthe oil-based adjuvant, VSA3 (U.S. Pat. No. 5,951,988, incorporatedherein by reference in its entirety; S. van Drunen Littel-van den Hurket al., Vaccine (1993) 11:25) such that each 2 ml dose contained 200 μgof CCS protein and 30% (v/v) of adjuvant (CCS vaccine). For the controlgroup, sterile saline was mixed with VSA3, such that each 2 ml dosecontained 0 μg of CCS protein and 30% (v/v) of adjuvant (salinevaccine).

Sixteen cattle were divided in 2 groups of eight animals each. Group 1cattle received 2 ml of CCS vaccine subcutaneously (experimental) andGroup 2 cattle received 2 ml saline vaccine subcutaneously (control) ondays 1 and 22 (boost). Seroconversion was assayed by ELISA (Example 3),on days 1 (pre-immunization), 22 and 36. As shown in Table 1, at day 22,Group 1 animals showed specific antibody titers to EspA and Tir and, atday 36, these titers showed a significant increase. Group 2 animalsshowed no specific antibody titers at days 22 and 36. In particular, thegroup which received the EHEC vaccine showed a 13-fold increase inspecific antibody titer to type III secreted proteins after a singleimmunization and following the first booster, the eight animals in theEHEC vaccine group demonstrated a 45-fold increase in specific antibodytiter while only one of the placebo vaccine group seroconverted (X²,p=0.0002).

TABLE 1 Serological response to immunization with CCS Specific AntibodyTiters*-Group Means Pre-immunization Boost Challenge Group (Day 1) (Day22) (Day 36) 1. Experimental 350 5,000 12,500 2. Control 450   500   650*Values are group means expressed as the reciprocal of the highestdilution yielding a positive result.

At day 36, Group 1 and Group 2 animals were challenged with 10⁸ CFU ofEHEC O157:H7 by oral-gastric intubation and fecal shedding was monitoredfor 14 days (Example 5). As summarized in Table 2, fewer experimentalanimals shed EHEC O157:H7 than control animals and experimental animalsthat did shed, shed EHEC O157:H7 for a shorter period of time thancontrol animals (FIG. 7). In particular, The median number of daysduring which the organism was shed in the vaccinated animals was 1.5compared to 3.5 in the placebo group (Wilcoxin Signed Rank Test,p=0.08). Seven out of eight placebo-immunized animals shed the bacteriaduring the trial and four of those animals shed the bacteria for four ormore consecutive days, indicating that they were persistently infected.Five out of eight EHEC vaccine-immunized animals shed bacteria at somepoint during the trial but only one animal shed the organism for morethan two consecutive days, indicating that colonization was transientand significantly less than the placebo group. The total number ofbacteria isolated from fecal samples was significantly lower among theEHEC-vaccinated group as compared to the placebo group (Wilcoxin SignedRank Test, p=0.05), with the former having a median of 6.25 colonyforming units (CFU) per gram of feces recovered compared to a medianvalue of 81.25 CFU/g for the latter. Thus, vaccination with the typeIII-secreted proteins appeared to reduce the ability of the organism tocolonize the intestine as reflected by the decrease in the number ofdays animals shed the organism as well as the numbers of shed bacteriadetected by fecal culture.

TABLE 2 Shedding by experimental and control animals ExperimentalControl Animals shedding >1 day 1/8 6/8 Number of days with scores of >11 8 Average days of shedding per animal 0.875 2.5 Total days sheddingper group 7 20

These data show that CCS induced an antibody response in cattle thatreduced both number of animals shedding EHEC O157:H7 and the number ofdays during which EHEC O157:H7 were shed.

In order to enhance the effectiveness of the vaccine formulation, groupsof 6 calves were immunized as described above with one of three doses ofsecreted proteins (50 μg, 100 μg, 200 μg) or a placebo and theserological response was measured in serum samples taken at days 0, 21(boost) and 35. No significant difference in anti-EHEC, anti-Tir oranti-EspA responses were observed between any of the groups whichreceived the EHEC vaccine at any time point but all three weresignificantly higher than the placebo group on days 21 and 35. Thus, asecond vaccine trial was designed in which three groups of yearlingcattle were immunized three times with 50 μg of secreted proteins(n=13), 50 μg of secreted proteins from a tir mutant (ΔTir, n=10) or aplacebo (n=25). The adjuvant used was VSA3 and animals were immunized bysubcutaneous injection on days 0, 21, and 35, followed by oral challengewith E. coli O157:H7 on day 49. The serological response to immunizationis shown in Table 3 (days 0 and 49 only) and was comparable to thatobserved in the trial described above. The group which received the ΔTirvaccine showed a response of similar magnitude against total secretedproteins as the group which received the vaccine prepared from thewild-type strain, but, as expected, a significantly reduced response toTir (Wilcoxin Signed Rank Test, p=0.006). However, the former group didshow an increase in anti-Tir antibody levels (Wilcoxin Signed Rank Test,p=0.009), indicating either exposure to an organism producing animmunologically related molecule or natural exposure to E. coli O157:H7.This is further supported by the observation that there was asignificant increase in the anti-Tir antibody titer in the placebo groupon the day of challenge (Wilcoxin Signed Rank Test, p=0.002) but nodifference between the placebo or ΔTir groups (p=0.37, Kruskal-WallisANOVA). The response to EspA was similar in both the EHEC and ΔTirvaccine groups (p=0.45, Kruskal-Wallis ANOVA) and was significantlyhigher than the placebo-immunized animals (p<0.0001).

TABLE 3 Median serological response to immunization with secretedproteins prepared from wild- type E. coli O157:H7 (EHEC), an isogenictir mutant (ΔTir) or a placebo. Titers are expressed as geometric meanvalues of the last positive dilution of sera ( ). Numbers in parenthesesrepresent the 25^(th)–75^(th) percentile. Anti-EHEC Anti-Tir Anti-EspAGroup n Day 0 Day 49 Day 0 Day 49 Day 0 Day 49 EHEC 13 10 6400 100 1600100 400 (10–100) (3200–12800) (10–200) (800–3200) (10–200) (200–1600)ΔTir 10 10 6400 10 200 100 300 (10–100) (3200–25600) (10–200) (100–800)(10–200) (100–1600) Placebo 25 10 10 100 200 100 100 (10–200) (10–200)(10–200) (10–400) (10–200) (10–200)

The immune response against each vaccine formulation was also analyzedqualitatively by Western blotting using sera from two representativeanimals per group. The results for representative animals are shown inFIG. 8 and demonstrate that the proteins secreted by the type III systemwere highly immunogenic in cattle. The response in the EHEC and ΔTirvaccine groups was similar with the exception of the response againstTir which was absent in the latter group (FIG. 8, top panels). EspB,EspD and Tir were all reactive, and following the second immunization onday 21 a significant response against lipopolysaccharide was alsoobserved. The kinetics of the immune response in a vaccinated animal(FIG. 8, bottom panels) show that anti-Tir antibodies were detectablefollowing a single immunization, as were antibodies against 43-kDa and100-kDa proteins. The latter proteins were produced by the wild-typestrain as well as the sepB and tir mutants and the 100 kDa protein isprobably EspP, a non-type III EHEC secreted protein.

Following oral challenge with E. coli O157:H7 on day 49, each group wasmonitored daily for fecal shedding of the organism for 14 days. In thisexperiment, bacteria were cultured following immunomagnetic enrichment(J. Van Donkersgoed et al., Can. Vet. J. (2001) 42:714, Chapman andSiddons, J. Med. Microbiol. (1996) 44:267) rather than direct platingsince yearling cattle shed less than calves in this infection model. Onthe day of challenge, two animals in the placebo group wereculture-positive for E. coli O157:H7 and were eliminated from the trial.The placebo-immunized animals shed the organism after challenge muchmore than those in the two EHEC vaccine groups (FIG. 9). Those whichreceived the placebo vaccine shed the organism for a median of 4 days,significantly longer than the median of 0 days by the other two vaccinegroups (p=0.0002, Kruskal-Wallis ANOVA). Significantly fewer bacteriawere recovered from the EHEC and ΔTir vaccine groups (p=0.04,Kruskal-Wallis ANOVA). From day 2 post-infection onwards, 78% of theplacebo animals shed the organism for at least one day as compared to15% of the EHEC and 30% of the ΔTir vaccinates (Table 4).

The data presented above demonstrate that virulence factors of EHEC,namely those secreted by the type III system, can be used as effectivevaccine components for the reduction of colonization of cattle by EHECbacteria, such as EHEC O157:H7. These proteins are major targets of theimmune response in humans following infection (Li et al., Infect. Immun.(2000) 68:5090), although cattle do not usually mount a significantserological response against these proteins following natural exposureto the organism. However, animals vaccinated with these proteins areprimed and show an increase in anti-EHEC and anti-Tir titers followingoral challenge with the organism.

Tir is likely required for colonization of the bovine intestine, andthis is supported by the observation that a vaccine containing secretedproteins from a ΔTir E. coli O157:H7 strain was not as efficacious as anidentical formulation from an isogenic wild-type isolate. However, theformer vaccine was significantly more efficacious than a placebosuggesting that immunity against colonization is multifactorial innature. This is supported by the Western blot analysis of the responseto immunization in which several protein components as well aslipopolysaccharide were recognized. The contribution to protection bylipopolysaccharide is not known, but the presence of antibodies againstthis molecule does not correlate with protection in a murine EHEC model(Conlan et al., Can. J. Microbiol. (1999) 45:279; Conlan et al., Can. J.Microbiol. (2000) 46:283). Also, immunization with recombinant Tir andEspA can reduce numbers of bacteria shed, but not the actual numbers ofanimals nor the duration of shedding.

The prevalence of non-O157 serotypes in North America appears to beincreasing and represents a significant portion of EHEC infections inother geographical locations. Since the type III-secreted antigensappear to be relatively conserved among non-O157 EHEC serotypes, thisvaccine formulation is likely broadly cross-protective, in contrast toformulations based upon the O157 LPS antigen.

TABLE 4 Number of animals shedding E. coli O157:H7 at any time betweenday 2 and day 14 post-challenge. Number Percent Vaccine Shedding nShedding p-value EHEC 2 13 15.4 0.003 ΔTir 3 10 30   0.008 Placebo 18 23 78.3 1   

EXAMPLE 7

Protective Capacity of rEspA+rTir and rEspB+rIntimin

rEspA, rTir, rEspB and rIntimin were mixed with the oil-based adjuvant,VSA3, such that each 2 ml dose contained 50 μg of rEspA+rTir or ofrEspB+rIntimin and 30% (v/v) of adjuvant. Sterile saline was mixed withVSA3, such that each 2 ml dose contained 0 μg of rEspA+rTir or ofrEspB+rIntimin and 30% (v/v) of adjuvant.

Thirty four cattle were divided in 4 groups. Ten cattle, Group 1, wereimmunized with rEspA+rTir vaccine (experimental) and 10 cattle, Group 2,were immunized with rEspB+rIntimin vaccine (experimental) on days 1, 22(boost) and 36. Seven cattle, Group 3, and 7 cattle, Group 4, wereimmunized with saline vaccine (control) an days 1, 22 (boost) and 36.Seroconversion was assayed by ELISA (Example 3) on days 1(pre-immunization), 22 and 36. As shown in FIG. 5, at day 22, Group 1animals showed specific antibody titers to rEspA and to rTir and Group 2animals showed specific antibody titers to rEspB and to rIntimin. Also,as shown in FIG. 5, at day 36, Group 1 animals showed an increase inspecific antibody titer to rTir and no change in specific antibody titerto rEspA and Group 2 animals showed an increase in specific antibodytiter to rIntimin and a decrease in specific antibody titer to rEspB.Groups 3 and 4 animals showed no specific antibody titers at days 22 and36.

At day 36, Groups 1–4 animals were challenged with 10⁸ CFU of EHECO157:H7 and shedding was monitored daily for 14 days (Example 5). Asshown in FIG. 6, differences in shedding between Group 1 (rTir+rEspA)animals and Group 3 (saline) animals was minimal during the first 5 dayspost-challenge. However, during the second week post-challengedifferences in Group 1 animals and Group 3 animals were evident. FewerGroup 1 animals shed EHEC O157:H7 than Group 3 animals. Group 1 animalsshed less EHEC O157:H7 in their feces for shorter time periods thanGroup 3 animals. Differences in shedding between Group 2(rEspB+rIntimin) and Group 4 (saline) animals were not evident withrespect to the number of animals shedding, the number of EHEC O157:H7shed and the time period of shedding.

These data show that the antibody response induced by rEspA+rTir vaccineinterfered with EHEC O157:H7 colonization of cattle, whereas theantibody response induced by rEspB+rIntimin vaccine did not interferewith EHEC O157:H7 colonization of cattle.

EXAMPLE 8

Protective Capacity of CCS+rEspA+rTir

CCS, CCS+rEspA, CCS+rTir, CCS+rEspA+rTir and saline are mixed with anadjuvant.

Twenty-five cattle are divided into 5 groups of five 5 cattle and areimmunized an days 1 and 22 (boost). Group 1 receives CCS vaccine, Group2 CCS+rEspA vaccine, Group 3 CCS+rTir vaccine, Group 4 CCS+rEspA+rTirvaccine, and Group 5 saline vaccine. Seroconversion is assayed by ELISA(Example 3) on days 1 (pre-immunization), 22 (boost) and 36. On days 22and 36 each of Groups 1–5 animals show specific antibody titers againstEspA and Tir, whereas Group 6 animals show no specific antibody titers.

At day 36, Groups 1–5 animals are challenged with 10⁸ CFU of EHECO157:H7 and shedding is monitored daily for 14 days (Example 5). Feweranimals in Groups 1–4 shed EHEC O157:H7 than animals in Group 5. Group 5animals shed the most EHEC O157:H7; Group 1 animals shed less EHECO157:H7 than Group 5 animals and Groups 2–4 animals shed less EHECO157:H7 than Group 1 animals.

EXAMPLE 9

Protective Capacity of CCS with Various Antigens

CSS is mixed with and adjuvant, such that each 2 ml dose contains 0, 50,100 or 200 μg of CCS and 30% (v/v) of adjuvant (Table 5).

TABLE 5 Protective capacity of CCS with various adjuvants Antigen Groupμg Adjuvant CCS 1  50 Emulsigen-Plus CCS 2 100 Emulsigen-Plus CCS 3 200Emulsigen-Plus CCS 4 200 Carbigen CCS 5 100 MCC CCS 6 200 MCC CCS 7 200MCC + Carbigen CCS 8 200 VSA CCS 9  0 Emulsigen-Plus (control)

Seventy-two cattle are divided in 9 groups of 8 cattle. Groups 1–8animals are immunized with CCS+adjuvant (Table 5) and Group 9 cattle areimmunized with saline+adjuvants on days 1 and 22 (boost). Seroconversionis assayed by ELISA (Example 3) on days 1 (pre-immunization), 22 (boost)and 36. Groups 1–8 (CCS+adjuvant) animals show specific antibody titersto EspA and Tir on days 22 and 36. Group 9 (saline+adjuvant) animalsshow no specific antibody titers on days 22 and 36.

EXAMPLE 10

Protective Capacity of CCS in Dairy Cows

Twenty adult dairy cows are divided in 2 groups of 10 cows. Group 1 isimmunized with CCS vaccine and Group 2 is immunized with saline-vaccineon days 1 and day 22 (boost). Seroconversion is assayed by ELISA(Example 3) on days 1 (pre-immunization), 22 and 36. On days 22 and 36Group 1 cows show specific antibody titers against EspA and Tir, whereasGroup 2 cows show no specific antibody titers.

At day 36, Groups 1 and 2 cows are challenged with 10⁸ CFU of EHECO157:H7 and shedding is monitored daily for 14 days (Example 5). FewerGroup 1 cows shed EHEC O157:H7 than Groups 2 cows. Group 1 cows shedless EHEC O157:H7 for a shorter period of time than Groups 2 cows.

Six months after the initial immunization, Group 1 and 2 cows are againimmunized (2nd boost) via the subcutaneous route. On day 14 followingthe 2nd boost, antibody titers are assayed by ELISA (Example 3). Group 1cows have specific antibody titers to EspA and Tir, whereas Group 2 cowshave no specific antibody titers.

On day 14 following the 2nd boost, Groups 1 and 2 cows are againchallenged with 10⁸ CFU of EHEC O157:H7 and shedding is monitored dailyfor 14 days (Example 5). Fewer Group 1 (CCS) cows shed EHEC O157:H7 thanGroup 2 (saline) cows. Group 1 cows shed less EHEC O157:H7 for a shortertime periods than Group 2 cows.

EXAMPLE 11

Protective Capacity of CCS in Calves

Ten weaned calves (3–6 month old) are divided into 2 groups of 5 calvesand are immunized prior to entry into a feed-lot (day 0) and on the dayof entry into a feed lot (day 1, boost). Group 1 calves receive CCSvaccine and Group 2 calves receive saline vaccine. Seroconversion isassayed by ELISA (Example 3) on days 0, 1 and 14. On days 1 and 14 Group1 (CCS) calves show specific antibody titers to EspA and Tir, whereasGroup 2 (saline) calves show no specific antibody titers.

At day 14, Groups 1 and 2 calves are challenged with 10⁸ CFU of EHECO157:H7 and shedding is assayed daily for 14 days (Example 5). FewerGroup 1 calves shed EHEC O157:H7 than Group 2 calves. Group 1 calvesshed less EHEC O157:H7 for a shorter time period than Group 2 calves.

Ten weaned calves (3–6 month old) we divided into 2 groups of 5 calvesand are immunized on the day of entry into a feed-lot (day 1) and on day22 (boost) in the feed lot. Group 1 calves receive CCS vaccine and Group2 calves receive saline vaccine. Seroconversion is assayed by ELISA(Example 3) on days 1 (pre-immunization), 22 and 36. On days 22 and 36Group 1 (CCS) calves show specific antibody titers to EspA and Tir,whereas Group 2 (saline) calves show no specific antibody titers.

At day 36, Groups 1 and 2 calves are challenged with 10⁸ CFU of EHECO157:H7 and shedding is assayed daily for 14 days (Example 5). FewerGroup 1 calves shed EHEC O157:H7 than Group 2 calves. Group 1 calvesshed less EHEC O157:H7 for a shorter time period than Group 2 calves.

EXAMPLE 12

Protective Capacity of CCS in Sheep

Twenty adult sheep are divided in 2 groups of 10 sheep. Group 1 isimmunized with CCS vaccine and Group 2 is immunized with saline vaccineon day 1 and day 22 (boost). Seroconversion is assayed by ELISA (Example3) on days 1 (pre-immunization), 22 and 36. On days 22 and 36 Group 1sheep show specific antibody titers against EspA and Tir, whereas Group2 sheep show no specific antibody titers.

At day 36, Groups 1 and 2 sheep are challenged with 10⁸ CFU of EHECO157:H7 and shedding is monitored daily for 14 days (Example 5). FewerGroup 1 sheep shed EHEC O157:H7 than Group 2 sheep. Group 1 sheep shedless EHEC O157:H7 for a shorter period of time than Group 2 sheep.

Thus, compositions and methods for treating and preventingenterohemorragic E. coli colonization of mammals have been disclosed.Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. A method for reducing colonization of enterohemorrhagic Escherichiacoli (EHEC) in a non-human mammal comprising administering to saidnon-human mammal an effective amount of a composition comprising anisolated EHEC cell culture supernatant, wherein the cell culturesupernatant is produced by the process of incubating the cell culture inmedia comprising minimal media supplemented with 0.1% Casamino Acids,0.4% glucose, 8 mM MgSO₄ and 44 mM NaHCO₃.
 2. A method for reducingshedding of enterohemorrhagic Escherichia coli (EHEC) from a non-humanmammal comprising administering to said non-human mammal an effectiveamount of a composition comprising an isolated EHEC cell culturesupernatant, wherein the cell culture supernatant is produced by theprocess of incubating the cell culture in media comprising minimal mediasupplemented with 0.1% Casamino Acids, 0.4% glucose, 8 mM MgSO₄ and 44mM NaHCO₃.
 3. The method of claim 1, wherein the non-human mammal is aruminant.
 4. The method of claim 3, wherein the ruminant is a bovinesubject.
 5. The method of claim 1, wherein the composition furthercomprises an immunological adjuvant.
 6. The method of claim 1, whereinthe EHEC is EHEC O157:H7.
 7. The method of claim 1, wherein the EHEC isEHEC O157:NM.
 8. The method of claim 5, wherein the immunologicaladjuvant comprises an oil-in-water emulsion.
 9. The method of claim 5,wherein the immunological adjuvant comprises a mineral oil anddimethyldioctadecylammonium bromide.
 10. The method of claim 5, whereinthe immunological adjuvant comprises a non-oil-in-water emulsion. 11.The method of claim 5, wherein the immunological adjuvant is present inthe composition at a concentration of 20% to 40% (v/v).
 12. The methodof claim 11, wherein the immunological adjuvant is present in thecomposition at a concentration of 30% (v/v).
 13. The method of claim 1,wherein the composition further comprises one or more recombinant orpurified EHEC antigens selected from the group consisting of EspA, EspB,EspD, Tir and Intimin.
 14. The method of claim 13, wherein EspA+Tircomprise 10% to 50% of EHEC protein in the cell culture supernatantpresent in the composition.
 15. The method of claim 5, wherein thecomposition further comprises one or more recombinant or purified EHECantigens selected from the group consisting of EspA, EspB, EspD, Tir andIntimin.
 16. The method of claim 15, wherein EspA+Tir comprise 10% to50% of EHEC protein in the cell culture supernatant present in thecomposition.
 17. The method of claim 2, wherein the non-human mammal isa ruminant.
 18. The method of claim 17, wherein the ruminant is a bovinesubject.
 19. The method of claim 2, wherein the composition furthercomprises an immunological adjuvant.
 20. The method of claim 2, whereinthe EHEC is EHEC O157:H7.
 21. The method of claim 2, wherein the EHEC isEHEC O157:NM.
 22. The method of claim 19, wherein the immunologicaladjuvant comprises an oil-in-water emulsion.
 23. The method of claim 19,wherein the immunological adjuvant comprises a mineral oil anddimethyldioctadecylammonium bromide.
 24. The method of claim 19, whereinthe immunological adjuvant comprises a non-oil-in-water emulsion. 25.The method of claim 19, wherein the immunological adjuvant is present inthe composition at a concentration of 20% to 40% (v/v).
 26. The methodof claim 19, wherein the immunological adjuvant is present in thecomposition at a concentration of 30% (v/v).
 27. The method of claim 2,wherein the composition further comprises one or more recombinant orpurified EHEC antigens selected from the group consisting of EspA, EspB,EspD, Tir and Intimin.
 28. The method of claim 27, wherein EspA+Tircomprise 10% to 50% of EHEC protein in the cell culture supernatantpresent in the composition.
 29. The method of claim 19, wherein thecomposition further comprises one or more recombinant or purified EHECantigens selected from the group consisting of EspA, EspB, EspD, Tir andIntimin.
 30. The method of claim 29, wherein EspA+Tir comprise 10% to50% of EHEC protein in the cell culture supernatant present in thecomposition.
 31. The method of claim 5 or 19, wherein the immunologicaladjuvant comprises an agent selected from the group consisting of anemulsifying agent, a muramyl dipeptide, an aqueous agent, achitosan-based agent, a saponin, an oil, a lipopolysaccharide, abacterial cell wall extract, a bacterial DNA, a bacterial complex, asynthetic oligonucleotide, and a aliphatic nitrogenous base.
 32. Themethod of claim 31, wherein the emulsifying agent is selected from thegroup consisting of a natural emulsifying agent, a synthetic emulsifyingagent, an anionic emulsifying agent, a cationic emulsifying agent, and anonionic agent.
 33. The method of claim 32, wherein the naturalemulsifying agent is selected from the group consisting of acacia,gelatin, lecithin, and cholesterol.
 34. The method of claim 32, whereinthe anionic emulsifying agent is selected from the group consisting of apotassium salt of lauric acid, a potassium salt of oleic acid, a sodiumsalt of lauric acid, a sodium salt of oleic acid, an ammonium salt oflauric acid, an ammonium salt of oleic acid, a calcium salt of a fattyacid, a magnesium salt of a fatty acid, an aluminum salt of a fattyacid, a metallic soap, and an organic sulfonate.
 35. The method of claim34, wherein the organic sulfonate is sodium lauryl sulfate.
 36. Themethod of claim 32, wherein the cationic emulsifying agent isceryltrimethylammonium bromide.
 37. The method of claim 32, wherein thesynthetic agent is selected from the group consisting of a glycerylester, a polyoxyethylene glycol ester, a polyoxyethylene glycol ether,and a sorbitan fatty acid ester.
 38. The method of claim 37, wherein theglyceryl ester is glyceryl monostearate.
 39. The method of claim 37,wherein the sorbitan fatty acid ester is selected from the groupconsisting of a sorbitan monopalmitate and polyoxyethylene derivativesthereof.
 40. The method of claim 37, wherein the polyoxyethylenederivatives is polyoxyethylene sorbitan monopalmitate.
 41. The method ofclaim 31, wherein the aqueous agent is aluminum hydroxide.
 42. Themethod of claim 31, wherein the oil is selected from the groupconsisting of a mineral oil, a vegetable oil, and an animal oil.
 43. Themethod of claim 42, wherein the vegetable oil is selected from the groupconsisting of canola oil, almond oil, cottonseed oil, corn oil, oliveoil, peanut oil, safflower oil, sesame oil, and soybean oil.
 44. Themethod of claim 42, wherein the animal oil is selected from the groupconsisting of cod liver oil, halibut oil, menhaden oil, orange roughyoil and shark liver oil.
 45. The method of claim 5 or 19, wherein theimmunological adjuvant comprises an oil component.
 46. The method ofclaim 45, wherein the oil component is selected from the groupconsisting of a single oil, and a mixture of oils.
 47. The method ofclaim 5 or 19, wherein the immunological adjuvant comprisesMycobacterial cell wall extract.
 48. The method of claim 5 or 19,wherein the immunological adjuvant comprises Mycobacterial DNA.
 49. Themethod of claim 5 or 19, wherein the immunological adjuvant comprises aMycobacterial cell wall complex.
 50. The method of claim 31, wherein thealiphatic nitrogenous base is selected from the group consisting of anamine, a quaternary ammonium compound, a guanidine, a benzamidine, and athiouronium.
 51. The method of claim 5 or 19, wherein the immunologicaladjuvant comprises dimethyl-dioctadecylammonium bromide.
 52. The methodof claim 31, wherein the aliphatic nitrogenous base isN,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine.
 53. The method ofclaim 14, 16, 18, or 30, wherein EspA+Tir comprise 20% EHEC protein inthe culture supernatant present in the composition.
 54. The method ofclaim 3 or 17, wherein the ruminant is an ovine subject.
 55. The methodof claim 19, wherein the immunological adjuvant comprises an agentselected from the group consisting of an emulsifying agent, a muramyldipeptide, an aqueous agent, a chitosan-based agent, a saponin, an oil,a lipopolysaccharide, a bacterial cell wall extract, a bacterial DNA, abacterial complex, a synthetic oligonucleotide, and a aliphaticnitrogenous base.
 56. The method of claim 19, wherein the immunologicaladjuvant comprises an oil component.
 57. The method of claim 24, whereinthe non-oil-in-water emulsion is selected from the group consisting ofan oil emulsion, a water-in-oil emulsion, and a water-in-oil-in-wateremulsion.
 58. The method of claim 19, wherein the immunological adjuvantcomprises Mycobacterial cell wall extract.
 59. The method of claim 19,wherein the immunological adjuvant comprises Mycobacterial DNA.
 60. Themethod of claim 19, wherein the immunological adjuvant comprises aMycobacterial cell wall complex.
 61. The method of claim 19, wherein theimmunological adjuvant comprises Mycobacterial cell wall extract. 62.The method of claim 19, wherein the immunological adjuvant comprisesMycobacterial DNA.
 63. The method of claim 19, wherein the immunologicaladjuvant comprises a Mycobacterial cell wall complex.
 64. The method ofclaim 19, wherein the immunological adjuvant comprisesdimethyl-dioctadecylammonium bromide.
 65. The method of claim 16,wherein EspA+Tir comprise 20% of EHEC protein in the cell culturesupernatant present in the composition.
 66. The method of claim 28,wherein EspA+Tir comprise 20% of EHEC protein in the cell culturesupernatant present in the composition.
 67. The method of claim 30,wherein EspA+Tir comprise 20% of EHEC protein in the cell culturesupernatant present in the composition.
 68. The method of claim 17,wherein the ruminant is an ovine subject.
 69. The method of claim 1,wherein the cell culture supernatant is concentrated.
 70. The method ofclaim 2, wherein the cell culture supernatant is concentrated.
 71. Amethod for reducing colonization of enterohemorrhagic Escherichia coli(EHEC) in a non-human mammal comprising administering to said non-humanmammal an effective amount of a composition comprising an isolated EHECcell culture supernatant containing one or more EHEC serotypes whereinthe cell culture supernatant is produced by the process of incubatingthe cell culture in media comprising minimal media supplemented with0.1% Casamino Acids, 0.4% glucose, 8 mM MgSO₄ and 44 mM NaHCO₃.
 72. Themethod of claim 71, wherein the cell culture supernatant isconcentrated.
 73. The method of claim 71, wherein the one of more EHECserotypes are selected from the group consisting of O157, O158, O5, O8,O18, O26, O45, O48, O52, O55, O75, O76, O78, O84, O91, O103, O104, O111,O113, O114, O116, O118, O119, O121, O125, O28, O145, O146, O163, andO165.
 74. The method of claim 71, wherein the step of administering isby a route selected from the group consisting of oral, subcutaneous,intramuscular, intravenous, subcutaneous, intradermal, transdermal andsubdermal.
 75. A method for reducing shedding of enterohemorrhagicEscherichia coli (EHEC) from a non-human mammal comprising administeringto said non-human mammal an effective amount of a composition comprisingan isolated EHEC cell culture supernatant containing one or more EHECserotypes wherein the cell culture supernatant is produced by theprocess of incubating the cell culture in media comprising minimal mediasupplemented with 0.1% Casamino Acids, 0.4% glucose, 8 mM MgSO₄ and 44mM NaHCO₃.
 76. The method of claim 75, wherein the cell culturesupernatant is concentrated.
 77. The method of claim 75, wherein the oneof more EHEC serotypes are selected from the group consisting of O157,O158, O5, O8, O18, O26, O45, O48, O52, O55, O75, O76, O78, O84, O91,O103, O104, O111, O113, O114, O116, O118, O119, O121, O125, O28, O145,O146, O163, and O165.
 78. The method of claim 75, wherein the step ofadministering is by a route selected from the group consisting of oral,subcutaneous, intramuscular, intravenous, subcutaneous, intradermal,transdermal and subdermal.
 79. The method of claim 1, wherein the stepof administering is by a route selected from the group consisting oforal, subcutaneous, intramuscular, intravenous, subcutaneous,intradermal, transdermal and subdermal.
 80. The method of claim 2,wherein the step of administering is by a route selected from the groupconsisting of oral, subcutaneous, intramuscular, intravenous,subcutaneous, intradermal, transdermal and subdermal.